Specific interactions between proteins and primary DNA sequences provide a basic framework through which numerous cellular functions are mediated: control of gene expression, initiation of DNA synthesis, genetic recombination, initiation of DNA repair and restriction/modification and compaction of nucleic acids. Within this framework of DNA-interactive proteins, two major categories of proteins can be distinguished as those which bind or modify DNA at very specific sequence arrays and those which interact with necleic acids through a more general mechanism in which they recognize common DNA structural features or DNA abnormalities. In contrast to the highly sequence-dependent protein-DNA associations (i.e. repressor molecules, polymerases), the DNA repair enzymes comprise a group of proteins which do not interact at specific arrays of DNA sequence but rather monitor DNA for aberrations which may have been caused by thermal, radiation or chemcial challenge or errors made during DNA replication. The lack of precise sequence recognition of these enzymes for their substrates does not minimize the requirement for precision in locating and recognizing the DNA aberration. Rather it mandates that the protein-DNA interactions occur through more generalized mechanisms and yet maintain a high degree of accuracy, even in the absence of invariant residue contacts. This proposal centers on the elucidation of the relationship between certain protein sequences which are found in one such DNA repair enzyme, the T4 endonuclease V and the observed biological and enzymatic functions of that protein. The determination of these more generalized structure-function relationships may serve as a working model from which extrapolations can be made to other DNA reactive proteins. This goal will be accomplished through the following specific aims: 1) an analysis of endonuclease V mutants which were produced by either oligonucleotide site-directed mutagenesis or region-specific mutagenesis of the cloned gene which codes for endonuclease V; the effects of those alterations on enzymatic functions both in vivo and in vitro will be determined, 2) structural comparisons between endonuclease V and a functionally related UV endonuclease, 3) footprinting studies to characterize specific points of contact between the protein and DNA, and 4) elucidation of the X-ray crystal structure for the T4 endonuclease V.